Protein hydrolysates and methods of making same

ABSTRACT

The present disclosure generally relates to methods of preparing protein hydrolysates for use in pediatric nutritional compositions. Specifically, alpha-, beta- and/or kappa casein enriched casein, acid casein or caseinates can be hydrolyzed with proteases such as trypsin (trypsin-like), chymotrypsin (chymotrypsin-like), pepsin and/or plasmin to produce a hydrolysate that is close to the peptide composition of human breast milk.

TECHNICAL FIELD

The present disclosure relates to methods of preparing proteinhydrolysates for use in pediatric nutritional compositions.

BACKGROUND

Exclusive breast feeding during the first six months after birth isrecommended by the World Health Organization (WHO), and efforts are madeto support and promote breast feeding amongst mothers worldwide.However, when a mother is unable to or chooses not to breast feed, it isnecessary to provide an infant with a suitable substitute nutritionalcomposition, such as an infant formula.

Accordingly, there is a need to provide nutritional compositions, suchas infant formulas, that provide a pediatric subject with proteincompositions close to those in human breast milk. The present disclosureaddresses this need by providing methods for producing hydrolysatecompositions that are more close to human breast milk than the proteinsources available in presently available pediatric nutritionalcompositions.

BRIEF SUMMARY

Applicants have determined the peptide composition of human breast milk(see, FIG. 1) and performed in silico analyses to predict whichproteases produced the peptides present in human milk (see, FIG. 2). Theanalysis of the peptide composition (i.e., “peptidome”) of human breastmilk has led to the discovery of methods for hydrolyzing non-human(e.g., bovine) protein sources using proteases as described herein, toprovide a peptide composition for use in a nutritional composition forpediatric subjects that are closer to human milk than prior artcompositions. Thus, the present disclosure relates in part to thepreparation of hydrolysates for use in pediatric nutritionalcompositions, e.g., an infant formula, wherein the hydrolysates areprepared so as to comprise a peptide composition close to that found inhuman milk.

In certain embodiments, the disclosure relates to a method for preparinga protein hydrolysate for use in a nutritional composition. The methodcan include hydrolyzing milk protein preparations such as a casein, acidcasein or caseinate using serine proteases such as trypsin (ortrypsin-like), chymotrypsin (or chymotrypsin-like) or plasmin, or anycombination thereof; or aspartyl proteases such as pepsin, or anycombination thereof. Trypsin-like refers to proteolytic enzymes havingproteolytic activity similar to that of trypsin, for example,proteolytic enzymes that cleave peptide bonds following a positivelycharged amino acid (e.g., lysine or arginine). Chymotrypsin-like refersto proteolytic enzymes having proteolytic activity similar to that ofchymotrypsin, for example, proteolytic enzymes that cleave peptide bondsfollowing a medium- to large-sized hydrophobic residue such as tyrosine,phenylalanine, and tryptophan.

In certain embodiments, trypsin, chymotrypsin and plasmin are used. Incertain embodiments, trypsin and chymotrypsin are used. In certainembodiments, trypsin and plasmin are used. In certain embodiments,chymotrypsin and plasmin are used. In certain embodiments, trypsin,chymotrypsin, plasmin and pepsin are used. In certain embodiments,trypsin, chymotrypsin and pepsin are used. In certain embodiments,trypsin, plasmin and pepsin are used. In certain embodiments,chymotrypsin, plasmin and pepsin are used. The proteases can be derivedfrom animal and/or microbial sources.

In certain embodiments, the milk protein preparation is a casein, acidcasein or caseinate, such as a beta-casein enriched casein, acid caseinor caseinate, an alpha-casein enriched casein, acid casein or caseinate,and/or a kappa-casein enriched casein, acid casein or caseinate. Incertain embodiments, the casein, acid casein or caseinate is beta-caseinenriched, and comprises between about 40-95% beta-casein. In certainembodiments, the casein, acid casein or caseinate is alpha-caseinenriched, and comprises between about 50-95% alpha-casein. In certainembodiments, the alpha-casein enriched casein, acid casein or caseinatefurther comprises between about 15-95% kappa-casein. In certainembodiments, the casein, acid casein or caseinate is kappa-caseinenriched and comprises between about 15-95% kappa-casein. In certainembodiments, the casein, acid casein or caseinate may contain wheyproteins in an amount no greater than 20%.

In another aspect, the disclosure relates to a method for preparing aprotein hydrolysate for use in a nutritional composition, the methodcomprising hydrolyzing polymeric immunoglobulin receptor (PIGR),osteopontin, bile-salt activated lipase and/or clusterin using aprotease or proteases. In certain embodiments, the protease is trypsin,chymotrypsin, pepsin and/or plasmin.

In certain embodiments, the method also entails including the proteinhydrolysate in a nutritional composition. The nutritional compositioncan be a pediatric nutritional composition such as an infant formula.The nutritional composition can include a protein source, a lipidsource, and/or a carbohydrate source. The nutritional composition canalso include a prebiotic and/or a probiotic. The probiotic can be aLactobacillus species, such as Lactobacillus rhamnosus GG. The probioticcan be non-viable or viable. The probiotic can be present in an amountranging from about 1×10⁵ cfu/100 kcals to about 1.5×10⁹ cfu/100 kcals.

In certain embodiments, the nutritional composition can include a humanmilk oligosaccharide (HMO), a long chain polyunsaturated fatty acid(e.g., docosahexaenoic acid and/or arachidonic acid), or a source ofβ-glucan (e.g., β-1,3-glucan).

In certain embodiments, the disclosure relates to a nutritionalcomposition comprising a protein hydrolysate as described herein.

It is to be understood that both the foregoing general description andthe following detailed description present embodiments of the disclosureand are intended to provide an overview or framework for understandingthe nature and character of the disclosure as it is claimed. Thedescription serves to explain the principles and operations of theclaimed subject matter. Other and further features and advantages of thepresent disclosure will be readily apparent to those skilled in the artupon a reading of the following disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a pie chart of human milk peptidome parent proteins. Theendogenous peptidome of human milk (n=27) was determined by LC-MS/MSbased peptidomics. The largest number of peptides originated fromcasein, in particular beta-casein.

FIG. 2 shows an enzyme prediction of the total human milk peptidome. Theendogenous peptidome of human milk (n=27) was determined by LC-MS/MSbased peptidomics and using EnzymePredictor software proteases werepredicted.

DETAILED DESCRIPTION

Reference now will be made in detail to the embodiments of the presentdisclosure, one or more examples of which are set forth herein below.Each example is provided by way of explanation of the nutritionalcomposition of the present disclosure and is not a limitation. In fact,it will be apparent to those skilled in the art that variousmodifications and variations can be made to the teachings of the presentdisclosure without departing from the scope or spirit of the disclosure.For instance, features illustrated or described as part of oneembodiment, can be used with another embodiment to yield a still furtherembodiment.

Thus, it is intended that the present disclosure covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents. Other objects, features and aspects of thepresent disclosure are disclosed in or are obvious from the followingdetailed description. It is to be understood by one of ordinary skill inthe art that the present discussion is a description of exemplaryembodiments only and is not intended as limiting the broader aspects ofthe present disclosure.

“Nutritional composition” means a substance or formulation thatsatisfies at least a portion of a pediatric subject's nutrientrequirements. The terms “nutritional(s),” “nutritional formula(s),”“enteral nutritional(s),” “nutritional composition(s),” and “nutritionalsupplement(s)” are used interchangeably throughout the presentdisclosure to refer to liquids, powders, gels, pastes, solids,concentrates, suspensions, or ready-to-use forms of enteral formulas,oral formulas, formulas for infants, formulas for pediatric subjects,formulas for children, growing-up milks and/or formulas for adults, suchas women who are lactating or pregnant. In particular embodiments, thenutritional compositions are for pediatric subjects, including infantsand children.

The term “enteral” means through or within the gastrointestinal, ordigestive, tract. “Enteral administration” includes oral feeding,intragastric feeding, transpyloric administration, or any otheradministration into the digestive tract.

“Pediatric subject” includes both infants and children, and refersherein to a human that is less than thirteen years of age. In someembodiments, a pediatric subject refers to a human subject that is lessthan eight years old. In other embodiments, a pediatric subject refersto a human subject between about one and about six years of age or aboutone and about three years of age. In still further embodiments, apediatric subject refers to a human subject between about 6 and about 12years of age.

“Infant” means a subject having an age of not more than about one yearand includes infants from about zero to about twelve months. The terminfant includes low birth weight infants, very low birth weight infants,and preterm infants. “Preterm” means an infant born before the end ofthe 37th week of gestation, while “full term” means an infant born afterthe end of the 37th week of gestation.

“Child” means a subject ranging in age from about twelve months to aboutthirteen years. In some embodiments, a child is a subject between theages of one and twelve years old. In other embodiments, the terms“children” or “child” refer to subjects that are between about one andabout six years old, between about one and about three years old, orbetween about seven and about twelve years old. In other embodiments,the terms “children” or “child” refer to any range of ages between about12 months and about 13 years.

“Children's nutritional product” refers to a composition that satisfiesat least a portion of the nutrient requirements of a child. A growing-upmilk is an example of a children's nutritional product.

“Infant formula” means a composition that satisfies at least a portionof the nutrient requirements of an infant. In the United States, thecontent of an infant formula is dictated by the federal regulations setforth at 21 C.F.R. Sections 100, 106, and 107. These regulations definemacronutrient, vitamin, mineral, and other ingredient levels in aneffort to simulate the nutritional and other properties of human breastmilk.

The term “growing-up milk” refers to a broad category of nutritionalcompositions intended to be used as a part of a diverse diet in order tosupport the normal growth and development of a child between the ages ofabout 1 and about 6 years of age.

“Milk-based” means comprising at least one component that has been drawnor extracted from the mammary gland of a mammal. In some embodiments, amilk-based nutritional composition comprises components of milk that arederived from domesticated ungulates, ruminants or other mammals or anycombination thereof. Moreover, in some embodiments, milk-based meanscomprising bovine casein, whey, lactose, or any combination thereof.Further, “milk-based nutritional composition” may refer to anycomposition comprising any milk-derived or milk-based product known inthe art.

“Nutritionally complete” means a composition that may be used as thesole source of nutrition, which would supply essentially all of therequired daily amounts of vitamins, minerals, and/or trace elements incombination with proteins, carbohydrates, and lipids. Indeed,“nutritionally complete” describes a nutritional composition thatprovides adequate amounts of carbohydrates, lipids, essential fattyacids, proteins, essential amino acids, conditionally essential aminoacids, vitamins, minerals and energy required to support normal growthand development of a subject.

Therefore, a nutritional composition that is “nutritionally complete”for a preterm infant will, by definition, provide qualitatively andquantitatively adequate amounts of carbohydrates, lipids, essentialfatty acids, proteins, essential amino acids, conditionally essentialamino acids, vitamins, minerals, and energy required for growth of thepreterm infant.

A nutritional composition that is “nutritionally complete” for a terminfant will, by definition, provide qualitatively and quantitativelyadequate amounts of all carbohydrates, lipids, essential fatty acids,proteins, essential amino acids, conditionally essential amino acids,vitamins, minerals, and energy required for growth of the term infant.

A nutritional composition that is “nutritionally complete” for a childwill, by definition, provide qualitatively and quantitatively adequateamounts of all carbohydrates, lipids, essential fatty acids, proteins,essential amino acids, conditionally essential amino acids, vitamins,minerals, and energy required for growth of a child.

As applied to nutrients, the term “essential” refers to any nutrientthat cannot be synthesized by the body in amounts sufficient for normalgrowth and to maintain health and that, therefore, must be supplied bythe diet. The term “conditionally essential” as applied to nutrientsmeans that the nutrient must be supplied by the diet under conditionswhen adequate amounts of the precursor compound is unavailable to thebody for endogenous synthesis to occur.

“Nutritional supplement” or “supplement” refers to a formulation thatcontains a nutritionally relevant amount of at least one nutrient. Forexample, supplements described herein may provide at least one nutrientfor a human subject, such as a lactating or pregnant female.

The term “protein equivalent” or “protein equivalent source” includesany protein source, such as soy, egg, whey, or casein, as well asnon-protein sources, such as peptides or amino acids. Further, theprotein equivalent source can be any used in the art, e.g., nonfat milk,whey protein, casein, soy protein, hydrolyzed protein, peptides, aminoacids, and the like. Bovine milk protein sources useful in practicingthe present disclosure include, but are not limited to, milk proteinpowders, milk protein concentrates, milk protein isolates, nonfat milksolids, nonfat milk, nonfat dry milk, whey protein, whey proteinisolates, whey protein concentrates, sweet whey, acid whey, casein, acidcasein, caseinate (e.g. sodium caseinate, sodium calcium caseinate,calcium caseinate), soy bean proteins, and any combinations thereof. Theprotein equivalent source can, in some embodiments comprise hydrolyzedprotein, including partially hydrolyzed protein and extensivelyhydrolyzed protein. The protein equivalent source may, in someembodiments, include intact protein.

The term “protein equivalent source” also encompasses free amino acids.In some embodiments, the amino acids may comprise, but are not limitedto, histidine, isoleucine, leucine, lysine, methionine, cysteine,phenylalanine, tyrosine, threonine, tryptophan, valine, alanine,arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine,proline, serine, carnitine, taurine and mixtures thereof. In someembodiments, the amino acids may be branched chain amino acids. Incertain other embodiments, small amino acid peptides may be included asthe protein component of the nutritional composition. Such small aminoacid peptides may be naturally occurring or synthesized.

The term “essential amino acid” as used herein refers to an amino acidthat cannot be synthesized de novo by the organism being considered orthat is produced in an insufficient amount, and therefore must besupplied by diet. For example, in some embodiments, where the targetsubject is a human, an essential amino acid is one that cannot besynthesized de novo by a human.

The term “non-essential amino acid” as used herein refers to an aminoacid that can be synthesized by the organism or derived by the organismfrom essential amino acids. For example, in some embodiments, where thetarget subject is a human, a non-essential amino acid is one that can besynthesized in the human body or derived in the human body fromessential amino acids.

“Probiotic” means a microorganism with low or no pathogenicity thatexerts at least one beneficial effect on the health of the host. Anexample of a probiotic is LGG.

In certain embodiments, the probiotic(s) may be viable or non-viable. Asused herein, the term “viable”, refers to live microorganisms. The term“non-viable” or “non-viable probiotic” means non-living probioticmicroorganisms, their cellular components and/or metabolites thereof.Such non-viable probiotics may have been heat-killed or otherwiseinactivated, but they retain the ability to favorably influence thehealth of the host. The probiotics useful in the present disclosure maybe naturally-occurring, synthetic or developed through the geneticmanipulation of organisms, whether such source is now known or laterdeveloped.

The term “non-viable probiotic” means a probiotic wherein the metabolicactivity or reproductive ability of the referenced probiotic has beenreduced or destroyed. More specifically, “non-viable” or “non-viableprobiotic” means non-living probiotic microorganisms, their cellularcomponents and/or metabolites thereof. Such non-viable probiotics mayhave been heat-killed or otherwise inactivated. The “non-viableprobiotic” does, however, still retain, at the cellular level, its cellstructure or other structure associated with the cell, for exampleexopolysaccharide and at least a portion its biological glycol-proteinand DNA/RNA structure and thus retains the ability to favorablyinfluence the health of the host. Contrariwise, the term “viable” refersto live microorganisms. As used herein, the term “non-viable” issynonymous with “inactivated”.

The term “equivalent” or “cell equivalent” refers to the level ofnon-viable, non-replicating probiotics equivalent to an equal number ofviable cells. The term “non-replicating” is to be understood as theamount of non-replicating microorganisms obtained from the same amountof replicating bacteria (cfu/g), including inactivated probiotics,fragments of DNA, cell wall or cytoplasmic compounds. In other words,the quantity of non-living, non-replicating organisms is expressed interms of cfu as if all the microorganisms were alive, regardless whetherthey are dead, non-replicating, inactivated, fragmented etc.

“Prebiotic” means a non-digestible food ingredient that beneficiallyaffects the host by selectively stimulating the growth and/or activityof one or a limited number of beneficial gut bacteria in the digestivetract, selective reduction in gut pathogens, or favorable influence ongut short chain fatty acid profile that can improve the health of thehost.

“β-glucan” means all β-glucan, including both β-1,3-glucan andβ-1,3;1,6-glucan, as each is a specific type of β-glucan. Moreover,β-1,3;1,6-glucan is a type of β-1,3-glucan. Therefore, the term“β-1,3-glucan” includes β-1,3;1,6-glucan.

All percentages, parts and ratios as used herein are by weight of thetotal formulation, unless otherwise specified.

The nutritional composition of the present disclosure may be free orsubstantially free of any optional or selected ingredients describedherein. In this context, and unless otherwise specified, the term“substantially free” means that the selected composition may containless than a functional amount of the optional ingredient, typically lessthan 0.1% by weight, and also, including zero percent by weight of suchoptional or selected ingredient. The compositions described herein maybe free or substantially free of any one or more of the followingcomponents: protein, lipid, GOS, PDX, prebiotics, LGG, probiotics, DHA,ARA, LCPUFAs, beta glucan (or any specific beta glucan describedherein), etc.

All references to singular characteristics or limitations of the presentdisclosure shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The compositions and methods of the present disclosure, includingcomponents thereof, can comprise, consist of, or consist essentially ofthe essential elements and limitations of the embodiments describedherein, as well as any additional or optional ingredients, components orlimitations described herein or otherwise useful in nutritionalcompositions.

As used herein, the term “about” should be construed to refer to both ofthe numbers specified in any range. Any reference to a range should beconsidered as providing support for any subset within that range.

Methods

The methods herein relate to the analysis of the peptide composition(i.e., “peptidome”) of human breast milk and discovery of methods forproviding a peptide composition for use in a nutritional composition forpediatric subjects that more closely mimics human milk. Thus, thepresent disclosure relates in part to the preparation of hydrolysatesfor use in pediatric nutritional compositions, e.g., an infant formula,wherein the hydrolysates are prepared so as to comprise a protein andpeptide-containing composition close to that found in human milk.

The methods disclosed herein relate, in part, to preparing hydrolysatesof beta-, alpha- or kappa-enriched casein, acid casein or caseinates,for use in, e.g., nutritional formulations. Casein refers to a family ofrelated phosphoproteins, including beta-casein, alpha-casein andkappa-casein. Bovine casein is commercially available from a variety ofsources. In certain embodiments, a casein that is enriched in beta-,alpha- or kappa-casein is used. Methods for enriching beta-casein (see,e.g., U.S. Patent Publication No. 20070104847) and alpha- andkappa-casein (see, e.g., WO2003003847) are known in the art. An acidcasein or a caseinate (e.g. sodium caseinate, sodium calcium caseinate,calcium caseinate) enriched in beta-, alpha-, or kappa-casein can alsobe used. Caseinates are typically formed by a reaction of acid caseinprotein with an alkali.

The methods disclosed herein further relate to preparing hydrolysates ofpolymeric immunoglobulin receptor (PIGR), osteopontin, bile-saltactivated lipase and/or clusterin with any one or more of the proteasesdescribed herein.

As described herein, hydrolysates can be made using one or moreproteases. Suitable proteases include trypsin, chymotrypsin, plasmin,pepsin, or any combination thereof. In certain embodiments, trypsin,chymotrypsin and plasmin are used. In certain embodiments, trypsin andchymotrypsin are used. In certain embodiments, trypsin and plasmin areused. In certain embodiments, chymotrypsin and plasmin are used. Incertain embodiments, trypsin, chymotrypsin, plasmin and pepsin are used.In certain embodiments, trypsin, chymotrypsin and pepsin are used. Incertain embodiments, trypsin, plasmin and pepsin are used. In certainembodiments, chymotrypsin, plasmin and pepsin are used. In certainembodiments, cathepsin D is also used (e.g., trypsin, chymotrypsin,plasmin, and cathepsin D are used; trypsin, chymotrypsin, and cathepsinD are used; trypsin, plasmin, and cathepsin D are used; chymotrypsin,plasmin, and cathepsin D are used; trypsin, chymotrypsin, plasmin,pepsin and cathepsin D are used; trypsin, chymotrypsin, pepsin andcathepsin D are used; trypsin, plasmin, pepsin and cathepsin D are used,chymotrypsin, plasmin, pepsin and cathepsin D are used). In certainembodiments, exonucleases are used. Proteases are known in the art andcan be obtained from any number of manufacturers, including, forexample, from Sigma Aldrich, St. Louis, Mo. and Worthington BiochemicalCorporation, Lakewood, N.J.

Methods for preparing casein hydrolysates are known in the art and aredescribed, for example, in Japanese Patent Application No. JP2006010357and in New Zealand Patent Application No. NZ619383, the disclosures ofwhich are hereby incorporated by reference for all purposes.

In certain embodiments, to prepare a hydrolysate, the protein (e.g., abeta-casein enriched casein) is dissolved or dispersed in a solvent suchas water (e.g., distilled water), which may include an acid or alkalineor their salts. The concentration of the solution can be between about1% and about 75% by weight, about 1% and about 50% by weight, about 1%and about 40% by weight, about 1% and about 30% by weight, about 1% andabout 20% by weight, about 1% and about 15% by weight, about 1 and about10% by weight, about 5% and about 15% by weight, about 5% and about 10%by weight.

The pH of the solution is then adjusted to within the operable range forthe protease or proteases to be used. Substrate concentration, enzymeconcentration, reaction temperature, reaction time, etc., are determinedfor the particular protease used. Reaction conditions for a given enzymeare known in the art and are typically provided by the manufacturer ofthe enzyme. For example, the pH range can be adjusted between pH 1 andpH 10, preferably in a range of 2-9. For some enzymes, pH is preferablyin a range of 6-9; whereas, pH for other enzymes is preferably in arange of 2-4. The pH can be adjusted during the process of enzymedigestion.

Progression of the reaction can be monitored by, for example, collectinga sample of the reaction solution at various time intervals, andmeasuring the extent of protein degradation, and optionally measuringmolecular weight distribution of the protein hydrolysates.

The reaction may be stopped by any means known in the art, for example,by addition of hydrochloride acid solution and/or heat inactivationtreatment. Heat deactivation treatment conditions (heating temperature,heating time, etc.), can be determined based upon the thermal stabilityof the enzyme used. The treatment can also combined with othertechnologies such as filtration, microfiltration, ultrafiltration, ornanofiltration to reduce and deactivate the enzyme proteins

After stopping the enzymatic reaction, the resulting hydrolysate may bepurified using one or more of filtration, microfiltration, membraneseparation processes such as ultrafiltration membrane, resin adsorptionseparation, from column chromatography. Membrane separation processescan be carried out using any apparatus known in the art. For example,microfiltration modules and ultrafiltration modules can be used tofilter the hydrolysate, which is obtained as a membrane permeantfraction. Resin adsorption separation can be carried out in any mannerknown in the art, for example, using resins, ion exchange resins,chelate resins, affinity adsorbent resin, a synthetic adsorbent, andhigh performance liquid chromatography resin.

Properties of the peptide hydrolysate can be tested and evaluated by,e.g., mass spectrometry and/or standard nitrogen and degree ofhydrolyses measurements. An exemplary mass spectrometer suitable for usewith the methods described herein is a high-performance liquidchromatograph triple quadrupole mass spectrometer (LC/MS/MS, WatersTQD). Hydrolysate can be separated by gradient analysis usingchromatography, e.g., a reverse phase ODS column as a separation columnand a 0.1% formic acid aqueous solution and 0.1% formic acid containingacetonitrile as eluents prior to measurement by mass spectrometer.Specific peptide content can be determined using a calibration curvewith a synthetic peptide as a standard and/or labeled peptide standards.

Nutritional Compositions

The protein hydrolysates prepared by the methods of the invention can beincluded in a nutritional composition, e.g., a pediatric nutritionalcomposition, individually, or in combination. In certain embodiments,the protein hydrolysate is present in an amount of about 0.036 g/100kcal to about 3 g/100 kcal of the nutritional composition, or about0.042 g/100 Kcal to about 2.5 g/100 Kcal, about 0.042 g/100 Kcal to 1.5g/100 Kcal, about 0.042 g/100 Kcal to about 1 g/100 Kcal, or about 0.042g/100 Kcal to about 0.5 g/100 Kcal. In certain embodiments, thehydrolysate is included in a nutritional composition as the sole sourceof protein. Additional components that can be added to the nutritionalcomposition are described below.

In certain embodiments, beta-casein peptides provide about 25% to about60% (e.g., about 30% to about 50%, about 35% to about 45%) of the totalpeptides in the nutritional composition. In certain embodiments,alpha-casein peptides provide about 5% to about 25% (e.g., about 10% toabout 20%, about 12% to about 18%) of the total peptides in thenutritional composition. In certain embodiments, PIGR peptides provideabout 5% to about 25% (e.g., about 10% to about 20%, about 12% to about18%) of the total peptides in the nutritional composition. In certainembodiments, osteopontin peptides provide about 1% to about 15% (e.g.,about 5% to about 10%, about 6% to about 8%) of the total peptides inthe nutritional composition. In certain embodiments, kappa-caseinpeptides provide about 1% to about 10% (e.g., about 2% to about 8%,about 3% to about 5%) of the total peptides in the nutritionalcomposition. In certain embodiments, bile salt-activated lipase peptidesprovide about 1% to about 10% (e.g., about 2% to about 8%, about 3% toabout 5%) of the total peptides in the nutritional composition. Incertain embodiments, clusterin peptides provide about 0.5% to about 5%(e.g., about 1% to about 3%, about 2%) of the total peptides in thenutritional composition.

The nutritional compositions described herein can also include a humanmilk oligosaccharide (HMO). The term “HMOs” or “human milkoligosaccharides” refers generally to a number of complex carbohydratesfound in human breast milk that can be in either acidic or neutral form.In certain embodiments, the HMO is 2′-fucosyllactose (2FL),3-fucosyllactose (3FL), 3′sialyllactose (3SL), 6′sialyllactose (6SL),lacto-N-biose (LNB), lacto-N-neotetraose (LnNT), lacto-N-tetraose (LNT),lacto-N-fucopentaose, lacto-N-fucopentaose I, lacto-N-fucopentaose II,lacto-N-fucopentaose III, lacto-N-fucopentaose V,lacto-N-neofucopentaose, lactodifucotetraose, lacto-N-difucohexaose II,lacto-N-neodifucohexaose II, para-lacto-N-neohexaose,3′sialyl-3fucosyllactose, sialy-lacto-N-tetraose, or any combinationthereof. 3′sialyllactose, 6′sialyllactose contribute sialic acid, whichis an important nutrient for brain development and cognitive function.Precursors of HMOs, such as sialic acid, fucose, N-acetylglucosamine, ora combination thereof, also may be included in the present compositions.

HMOs may be isolated or enriched from milk or produced via microbialfermentation, enzymatic processes, chemical synthesis, or a combinationthereof. For example, the HMOs disclosed herein may be derived from cowmilk, cow colostrum, goat milk, goat colostrum, horse milk, horsecolostrum, or any combination thereof.

HMOs are believed to correlate with the presence of beneficial infantspecific Bifidobacterium species, such as B. longum, B. infantis, B.breve, and B. bifidium in breast fed infants. Accordingly, thecompositions and methods described herein can be useful in increasing ormaintaining the amount of one or more of B. longum, B. infantis, B.breve, and B. bifidium in the gastrointestinal tract (e.g., gut) of thepediatric subject. In certain embodiments, providing to a pediatricsubject HMOs similar to those in the mother's breast milk can affect thecomposition of gastrointestinal bacteria in the pediatric subject, andmake the composition of gastrointestinal bacteria more similar to thatwhich would occur if the pediatric subject consumed his mother's breastmilk.

The HMOs, in certain embodiments, is present in the compositions in anamount ranging from about 0.005 g/100 kcal to about 1 g/100 kcal. Inother embodiments, the HMOs may be present in an amount ranging fromabout 0.01 g/100 kcal to about 1 g/100 kcal, about 0.02 g/100 kcal toabout 1 g/100 kcal, about 0.3 g to about 1 g/100 kcal, about 0.1 g/100kcal to about 0.8 g/100 kcal, or about 0.1 g/100 kcal to about 0.5 g/100kcal.

In some embodiments the nutritional compositions include from about 0.01g/100 kcal to about 0.8 g/100 kcal of sialylated HMOs. In otherembodiments, the nutritional compositions include from about 0.03 g/100kcal to about 0.6 g/100 kcal of sialylated HMOs. Still in someembodiments, then nutritional compositions include from about 0.04 g/100kcal to about 0.8 g/100 kcal of sialylated HMOs. Still in otherembodiments, the nutritional compositions include from about 0.05 g/100kcal to about 0.6 g/100 kcal of sialylated HMOs.

In some embodiments, the nutritional compositions include from about0.01 g/100 kcal to about 0.2 g/100 kcal of fucosylated HMOs. In someembodiments, the nutritional compositions include from about 0.02 g/100kcal to about 0.2 g/100 kcal of fucosylated HMOs. In some embodiments,the nutritional compositions include from about 0.05 g/100 kcal to about0.1 g/100 kcal of fucosylated HMOs.

In some embodiments, the nutritional compositions include from about0.01 g/100 kcal to about 0.5 g/100 kcal of HMOs that are neithersialyated nor fucosylated. In certain embodiments, the nutritionalcompositions include from about 0.025 g/100 kcal to about 0.5 g/100 kcalof HMOs that are neither sialylated nor fucosylated. In otherembodiments, the nutritional compositions contain from about 0.25 g/100kcal to about 0.7 g/100 kcal of HMOs that are neither sialylated norfucosylated. Indeed, in certain embodiments, the majority of the HMOsincluded in the nutritional compositions are neither sialylated norfucosylated.

In some embodiments, the nutritional composition may be formulated toinclude a certain weight percentage of HMOs based on the total amount ofcarbohydrates present in the nutritional compositions. Accordingly, insome embodiments the nutritional composition may include from about 0.1wt % to about 25 wt % HMOs based on the total weight of carbohydrates inthe nutritional composition. In some embodiments, the nutritionalcomposition includes from about 0.5 wt % to about 25 wt % HMOs based onthe total weight of carbohydrates in the nutritional composition. Insome embodiments, the nutritional composition includes from about 1 wt %to about 25 wt % HMOs based on the total weight of carbohydrates in thenutritional composition. In some embodiments, the nutritionalcomposition includes from about 2 wt % to about 20 wt % HMOs based onthe total weight of the carbohydrates in the nutritional composition.Still in some embodiments, the nutritional composition includes fromabout 5 wt % to about 15 wt % HMOs based on the total weight of thecarbohydrates in the nutritional composition. In some embodiments, thenutritional composition includes from about 8 wt % to about 12 wt % HMOsbased on the total weight of the carbohydrates in the nutritionalcomposition. Still, in certain embodiments, the nutritional compositionis formulated to include from about 0.1 wt % to about 5 wt % of HMOsbased on the total weight of the carbohydrates in the nutritionalcomposition.

Precursors of HMOs, such as sialic acid, fucose, N-acetylglucosamine, ora combination thereof, also may be included in the present compositions.

The nutritional composition may also contain one or more prebiotics(also referred to as a prebiotic source), in addition to HMOs, incertain embodiments. Prebiotics can stimulate the growth and/or activityof ingested probiotic microorganisms, selectively reduce pathogens foundin the gut, and favorably influence the short chain fatty acid profileof the gut. Such prebiotics may be naturally-occurring, synthetic, ordeveloped through the genetic manipulation of organisms and/or plants,whether such new source is now known or developed later. Prebioticsuseful in the present disclosure may include oligosaccharides,polysaccharides, and other prebiotics that contain fructose, xylose,soya, galactose, glucose and mannose.

More specifically, prebiotics useful in the present disclosure mayinclude polydextrose, polydextrose powder, lactulose, lactosucrose,raffinose, gluco-oligosaccharide, inulin, fructo-oligosaccharide,isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose,xylo-oligosaccharide, chito-oligosaccharide, manno-oligosaccharide,aribino-oligosaccharide, sialyl-oligosaccharide, fuco-oligosaccharide,galacto-oligosaccharide, and gentio-oligosaccharides. In someembodiments, the total amount of prebiotics present in the nutritionalcomposition may be from about 0.1 g/100 kcal to about 1.5 g/100 kcal. Incertain embodiments, the total amount of prebiotics present in thenutritional composition may be from about 0.3 g/100 kcal to about 1.0g/100 kcal. Moreover, the nutritional composition may comprise aprebiotic component comprising polydextrose (“PDX”) and/orgalacto-oligosaccharide (“GOS”). In some embodiments, the prebioticcomponent comprises at least 20% GOS, PDX or a mixture thereof.

In certain embodiments, the HMO component may be included in combinationwith GOS and PDX. The nutritional composition can comprise from about0.1 g/100 kcal to about 5 g/100 kcal of prebiotics, including GOS, PDX,and HMOs. Still in certain embodiments, the nutritional composition caninclude from about 0.1 g/100 kcal to about 4 g/100 kcal of prebiotics,including GOS, PDX, and HMOs.

In certain embodiments, the prebiotic component comprises both GOS andPDX. The GOS and PDX may be present in a ratio of about 1:9 to about 9:1by weight. In other embodiments, the GOS and PDX are present in a ratioof about 1:4 to 4:1, or about 1:1. In another embodiment, the ratio ofPDX:GOS can be between about 5:1 and 1:5. In yet another embodiment, theratio of PDX:GOS can be between about 1:3 and 3:1. In a particularembodiment, the ratio of PDX to GOS can be about 5:5. In anotherparticular embodiment, the ratio of PDX to GOS can be about 8:2.

In some embodiments, the amount of GOS in the nutritional compositionmay be from about 0.1 g/100 kcal to about 1.0 g/100 kcal. In anotherembodiment, the amount of GOS in the nutritional composition may be fromabout 0.1 g/100 kcal to about 0.5 g/100 kcal. The amount of PDX in thenutritional composition may, in some embodiments, be within the range offrom about 0.1 g/100 kcal to about 0.5 g/100 kcal. In other embodiments,the amount of PDX may be about 0.3 g/100 kcal.

In a particular embodiment, GOS and PDX are supplemented into thenutritional composition in a total amount of about at least about 0.2g/100 kcal and can be about 0.2 g/100 kcal to about 1.5 g/100 kcal. Insome embodiments, the nutritional composition may comprise GOS and PDXin a total amount of from about 0.6 to about 0.8 g/100 kcal.

In some embodiments the nutritional composition comprises a probiotic,and more particularly, Lactobacillus rhamnosus GG (LGG, ATCC number53103). Other probiotics useful in the present nutritional compositionsinclude, but are not limited to, Bifidobacterium species (e.g.,Bifidobacterium animalis, Bifidobacterium breve B-3, and Bifidobacteriumlongum subsp. infantis M-63), and combinations thereof.

In certain embodiments, one or more of the probiotics can be present inthe nutritional composition in an amount of from about 1×10⁴ cfu/100kcal to about 1.5×10¹⁰ cfu/100 kcal or about 1×10⁴ cell equivalent/100kcal to about 1.5×10¹⁰ cell equivalent/100 kcal. In other embodiments,the nutritional composition comprises one or more of the probiotics inan amount of from about 1×10⁶ cfu/100 kcal to about 1×10⁹ cfu/100 kcalor about 1×10⁶ cell equivalent/100 kcal to about 1.5×10⁹ cellequivalent/100 kcal. Still, in certain embodiments, the nutritionalcomposition may include one or more of the probiotics in an amount offrom about 1×10⁷ cfu/100 kcal to about 1×10⁸ cfu/100 kcal or about 1×10⁷cell equivalent/100 kcal to about 1.5×10⁸ cell equivalent/100 kcal. Theprobiotic may be either non-viable or viable.

In some embodiments, rather than (or in addition to) adding a probioticto the composition, probiotic functionality is provided by including aculture supernatant from a late-exponential growth phase of a probioticbatch-cultivation process, as disclosed in international publishedapplication no. WO 2013/142403, which is hereby incorporated byreference in its entirety. Without wishing to be bound by theory, it isbelieved that the activity of the culture supernatant can be attributedto the mixture of components (including proteinaceous materials, andpossibly including (exo)polysaccharide materials) released into theculture medium at a late stage of the exponential (or “log”) phase ofbatch cultivation of the probiotic. The term “culture supernatant” asused herein, includes the mixture of components found in the culturemedium. The stages recognized in batch cultivation of bacteria are knownto the skilled person. These are the “lag,” the “log” (“logarithmic” or“exponential”), the “stationary” and the “death” (or “logarithmicdecline”) phases. In all phases during which live bacteria are present,the bacteria metabolize nutrients from the media, and secrete (exert,release) materials into the culture medium. The composition of thesecreted material at a given point in time of the growth stages is notgenerally predictable.

In an embodiment, a culture supernatant is obtainable by a processcomprising the steps of (a) subjecting a probiotic such as LGG tocultivation in a suitable culture medium using a batch process; (b)harvesting the culture supernatant at a late exponential growth phase ofthe cultivation step, which phase is defined with reference to thesecond half of the time between the lag phase and the stationary phaseof the batch-cultivation process; (c) optionally removing low molecularweight constituents from the supernatant so as to retain molecularweight constituents above 5-6 kiloDaltons (kDa); (d) removing liquidcontents from the culture supernatant so as to obtain the composition.

The culture supernatant may comprise secreted materials that areharvested from a late exponential phase. The late exponential phaseoccurs in time after the mid exponential phase (which is halftime of theduration of the exponential phase, hence the reference to the lateexponential phase as being the second half of the time between the lagphase and the stationary phase). In particular, the term “lateexponential phase” is used herein with reference to the latter quarterportion of the time between the lag phase and the stationary phase ofthe probiotic (e.g., LGG) batch-cultivation process. In someembodiments, the culture supernatant is harvested at a point in time of75% to 85% of the duration of the exponential phase, and may beharvested at about ⅚ of the time elapsed in the exponential phase.

In some embodiments, a soluble mediator preparation is prepared from theculture supernatant as described below. Furthermore, preparation of anLGG soluble mediator preparation is described in US 20130251829 and US20110217402, each of which is incorporated by reference in its entirety.The stages recognized in batch cultivation of bacteria are known to theskilled person. These are the “lag,” the “log” (“logarithmic” or“exponential”), the “stationary” and the “death” (or “logarithmicdecline”) phases. In all phases during which live bacteria are present,the bacteria metabolize nutrients from the media, and secrete (exert,release) materials into the culture medium. The composition of thesecreted material at a given point in time of the growth stages is notgenerally predictable.

In certain embodiments, the soluble mediator preparation is obtainableby a process comprising the steps of (a) subjecting a probiotic such asLGG to cultivation in a suitable culture medium using a batch process;(b) harvesting a culture supernatant at a late exponential growth phaseof the cultivation step, which phase is defined with reference to thesecond half of the time between the lag phase and the stationary phaseof the batch-cultivation process; (c) optionally removing low molecularweight constituents from the supernatant so as to retain molecularweight constituents above 5-6 kiloDaltons (kDa); (d) removal of anyremaining cells using 0.22 μm sterile filtration to provide the solublemediator preparation; (e) removing liquid contents from the solublemediator preparation so as to obtain the composition.

In certain embodiments, secreted materials are harvested from a lateexponential phase. The late exponential phase occurs in time after themid exponential phase (which is halftime of the duration of theexponential phase, hence the reference to the late exponential phase asbeing the second half of the time between the lag phase and thestationary phase). In particular, the term “late exponential phase” isused herein with reference to the latter quarter portion of the timebetween the lag phase and the stationary phase of the LGGbatch-cultivation process. In a preferred embodiment of the presentdisclosure and embodiments thereof, harvesting of the culturesupernatant is at a point in time of 75% to 85% of the duration of theexponential phase, and most preferably is at about ⅚ of the time elapsedin the exponential phase.

The term “cultivation” or “culturing” refers to the propagation ofmicro-organisms, in this case LGG, on or in a suitable medium. Such aculture medium can be of a variety of kinds, and is particularly aliquid broth, as customary in the art. A preferred broth, e.g., is MRSbroth as generally used for the cultivation of lactobacilli. MRS brothgenerally comprises polysorbate, acetate, magnesium and manganese, whichare known to act as special growth factors for lactobacilli, as well asa rich nutrient base. A typical composition comprises (amounts ing/liter): peptone from casein 10.0; meat extract 8.0; yeast extract 4.0;D(+)-glucose 20.0; dipotassium hydrogen phosphate 2.0; Tween® 80 1.0;triammonium citrate 2.0; sodium acetate 5.0; magnesium sulfate 0.2;manganese sulfate 0.04.

In certain embodiments, the soluble mediator preparation is incorporatedinto an infant formula or other nutritional composition. The harvestingof secreted bacterial products brings about a problem that the culturemedia cannot easily be deprived of undesired components. Thisspecifically relates to nutritional products for relatively vulnerablesubjects, such as infant formula or clinical nutrition. This problem isnot incurred if specific components from a culture supernatant are firstisolated, purified, and then applied in a nutritional product. However,it is desired to make use of a more complete culture supernatant. Thiswould serve to provide a soluble mediator composition better reflectingthe natural action of the probiotic (e.g. LGG).

Accordingly, it is desired to ensure that the composition harvested fromLGG cultivation does not contain components (as may present in theculture medium) that are not desired, or generally accepted, in suchformula. With reference to polysorbate regularly present in MRS broth,media for the culturing of bacteria may include an emulsifying non-ionicsurfactant, e.g. on the basis of polyethoxylated sorbitan and oleic acid(typically available as Tween® polysorbates, such as Tween® 80). Whilstthese surfactants are frequently found in food products, e.g. ice cream,and are generally recognized as safe, they are not in all jurisdictionsconsidered desirable, or even acceptable for use in nutritional productsfor relatively vulnerable subjects, such as infant formula or clinicalnutrition.

Therefore, in some embodiments, a preferred culture medium of thedisclosure is devoid of polysorbates such as Tween 80. In a preferredembodiment of the disclosure and/or embodiments thereof the culturemedium may comprise an oily ingredient selected from the groupconsisting of oleic acid, linseed oil, olive oil, rape seed oil,sunflower oil and mixtures thereof. It will be understood that the fullbenefit of the oily ingredient is attained if the presence of apolysorbate surfactant is essentially or entirely avoided.

More particularly, in certain embodiments, an MRS medium is devoid ofpolysorbates. Also preferably medium comprises, in addition to one ormore of the foregoing oils, peptone (typically 0-10 g/L, especially0.1-10 g/L), meat extract (typically 0-8 g/L, especially 0.1-8 g/L),yeast extract (typically 4-50 g/L), D(+) glucose (typically 20-70 g/L),dipotassium hydrogen phosphate (typically 2-4 g/L), sodium acetatetrihydrate (typically 4-5 g/L), triammonium citrate (typically 2-4 g/L),magnesium sulfate heptahydrate (typically 0.2-0.4 g/L) and/or manganoussulfate tetrahydrate (typically 0.05-0.08 g/L).

The culturing is generally performed at a temperature of 20° C. to 45°C., more particularly at 35° C. to 40° C., and more particularly at 37°C. In some embodiments, the culture has a neutral pH, such as a pH ofbetween pH 5 and pH 7, preferably pH 6.

In some embodiments, the time point during cultivation for harvestingthe culture supernatant, i.e., in the aforementioned late exponentialphase, can be determined, e.g. based on the OD600 nm and glucoseconcentration. OD600 refers to the optical density at 600 nm, which is aknown density measurement that directly correlates with the bacterialconcentration in the culture medium.

The culture supernatant can be harvested by any known technique for theseparation of culture supernatant from a bacterial culture. Suchtechniques are known in the art and include, e.g., centrifugation,filtration, sedimentation, and the like. In some embodiments, LGG cellsare removed from the culture supernatant using 0.22 μm sterilefiltration in order to produce the soluble mediator preparation. Theprobiotic soluble mediator preparation thus obtained may be usedimmediately, or be stored for future use. In the latter case, theprobiotic soluble mediator preparation will generally be refrigerated,frozen or lyophilized. The probiotic soluble mediator preparation may beconcentrated or diluted, as desired.

The soluble mediator preparation is believed to contain a mixture ofamino acids, oligo- and polypeptides, and proteins, of various molecularweights. The composition is further believed to contain polysaccharidestructures and/or nucleotides.

In some embodiments, the soluble mediator preparation of the presentdisclosure excludes lower molecular weight components, generally below 6kDa, or even below 5 kDa. In these and other embodiments, the solublemediator preparation does not include lactic acid and/or lactate salts.These lower molecular weight components can be removed, for example, byfiltration or column chromatography. In some embodiments, the culturesupernatant is subjected to ultrafiltration with a 5 kDa membrane inorder to retain constituents over 5 kDa. In other embodiments, theculture supernatant is desalted using column chromatography to retainconstituents over 6 kDa.

The soluble mediator preparation of the present disclosure can beformulated in various ways for administration to pediatric subjects. Forexample, the soluble mediator preparation can be used as such, e.g.incorporated into capsules for oral administration, or in a liquidnutritional composition such as a drink, or it can be processed beforefurther use. Such processing generally involves separating the compoundsfrom the generally liquid continuous phase of the supernatant. Thispreferably is done by a drying method, such as spray-drying orfreeze-drying (lyophilization). In a preferred embodiment of thespray-drying method, a carrier material will be added beforespray-drying, e.g., maltodextrin DE29.

Probiotic bacteria soluble mediator preparations, such as the LGGsoluble mediator preparation disclosed herein, advantageously possessgut barrier enhancing activity by promoting gut barrier regeneration,gut barrier maturation and/or adaptation, gut barrier resistance and/orgut barrier function. The present LGG soluble mediator preparation mayaccordingly be particularly useful in treating subjects, particularlypediatric subjects, with impaired gut barrier function, such as shortbowel syndrome or NEC. The soluble mediator preparation may beparticularly useful for infants and premature infants having impairedgut barrier function and/or short bowel syndrome.

Probiotic bacteria soluble mediator preparation, such as the LGG solublemediator preparation of the present disclosure, also advantageouslyreduce visceral pain sensitivity in subjects, particularly pediatricsubjects experiencing gastrointestinal pain, food intolerance, allergicor non-allergic inflammation, colic, IBS, and infections.

The nutritional composition of the disclosure may contain a source oflong chain polyunsaturated fatty acid (LCPUFA), e.g., docosahexaenoicacid (DHA). Other suitable LCPUFAs include, but are not limited to,linoleic (18:2 n-6), γ-linolenic (18:3 n-6), dihomo-γ-linolenic (20:3n-6) acids in the n-6 pathway, α-linolenic (18:3 n-3), stearidonic (18:4n-3), eicosatetraenoic (20:4 n-3), eicosapentaenoic (20:5 n-3), anddocosapentaenoic (22:6 n-3) and arachidonic acid (ARA).

In certain embodiments the amount of LCPUFA in the nutritionalcomposition is at least about 5 mg/100 Kcal, and may vary from about 5mg/100 Kcal to about 100 mg/100 Kcal, more preferably from about 10mg/100 Kcal to about 50 mg/100 Kcal.

In certain embodiments, the amount of DHA in the nutritional compositionis at least about 17 mg/100 Kcal, and can vary from about 5 mg/100 Kcalto about 75 mg/100 Kcal, or from about 10 mg/100 Kcal to about 50 mg/100Kcal.

In an embodiment, especially if the nutritional composition is an infantformula, the nutritional composition is supplemented with both DHA andARA. In this embodiment, the weight ratio of ARA:DHA may be betweenabout 1:3 and about 9:1. In a particular embodiment, the ratio ofARA:DHA is from about 1:2 to about 4:1.

If included, the source of DHA and/or ARA may be any source known in theart such as marine oil, fish oil, single cell oil, egg yolk lipid, andbrain lipid. In some embodiments, the DHA and ARA are sourced fromsingle cell Martek oils, DHASCO® and ARASCO®, or variations thereof. TheDHA and ARA can be in natural form, provided that the remainder of theLCPUFA source does not result in any substantial deleterious effect onthe subject. Alternatively, the DHA and ARA can be used in refined form.

In an embodiment, sources of DHA and ARA are single cell oils as taughtin U.S. Pat. Nos. 5,374,657; 5,550,156; and 5,397,591, the disclosuresof which are incorporated herein in their entirety by reference.Nevertheless, the present disclosure is not limited to only such oils.

The nutritional composition may also comprise a source of beta-glucan.Glucans are polysaccharides, specifically polymers of glucose, which arenaturally occurring and may be found in cell walls of bacteria, yeast,fungi, and plants. Beta glucans (β-glucans) are themselves a diversesubset of glucose polymers, which are made up of chains of glucosemonomers linked together via beta-type glycosidic bonds to form complexcarbohydrates.

β-1,3-glucans are carbohydrate polymers purified from, for example,yeast, mushroom, bacteria, algae, or cereals. The chemical structure ofβ-1,3-glucan depends on the source of the β-1,3-glucan. Moreover,various physiochemical parameters, such as solubility, primarystructure, molecular weight, and branching, play a role in biologicalactivities of β-1,3-glucans. (Yadomae T., Structure and biologicalactivities of fungal beta-1,3-glucans. Yakugaku Zasshi. 2000;120:413-431.)

β-1,3-glucans are naturally occurring polysaccharides, with or withoutβ-1,6-glucose side chains that are found in the cell walls of a varietyof plants, yeasts, fungi and bacteria. β-1,3;1,6-glucans are thosecontaining glucose units with (1,3) links having side chains attached atthe (1,6) position(s). β-1,3;1,6 glucans are a heterogeneous group ofglucose polymers that share structural commonalities, including abackbone of straight chain glucose units linked by a β-1,3 bond withβ-1,6-linked glucose branches extending from this backbone. While thisis the basic structure for the presently described class of β-glucans,some variations may exist. For example, certain yeast β-glucans haveadditional regions of β(1,3) branching extending from the β(1,6)branches, which add further complexity to their respective structures.

β-glucans derived from baker's yeast, Saccharomyces cerevisiae, are madeup of chains of D-glucose molecules connected at the 1 and 3 positions,having side chains of glucose attached at the 1 and 6 positions.Yeast-derived β-glucan is an insoluble, fiber-like, complex sugar havingthe general structure of a linear chain of glucose units with a β-1,3backbone interspersed with β-1,6 side chains that are generally 6-8glucose units in length. More specifically, β-glucan derived frombaker's yeast is poly-(1,6)-β-D-glucopyranosyl-(1,3)-β-D-glucopyranose.

Furthermore, β-glucans are well tolerated and do not produce or causeexcess gas, abdominal distension, bloating or diarrhea in pediatricsubjects. Addition of β-glucan to a nutritional composition for apediatric subject, such as an infant formula, a growing-up milk oranother children's nutritional product, will improve the pediatricsubject's immune response by increasing resistance against invadingpathogens and therefore maintaining or improving overall health.

In some embodiments, the amount of β-glucan in the nutritionalcomposition is between about 3 mg and about 17 mg per 100 Kcal. Inanother embodiment the amount of β-glucan is between about 6 mg andabout 17 mg per 100 Kcal.

In a particular embodiment, a nutritional composition comprises per 100kcal: (i) between about 1 g and about 7 g of a protein source, (ii)between about 1 g and about 10 g of a lipid source, (iii) between about6 g and about 22 g of a carbohydrate source, (iv) between about 0.005 gand about 1 g of a human milk oligosaccharide, (v) between about 0.1 gand 1.0 g of a galacto-oligosaccharide, (vi) between about 0.1 g andabout 0.5 g of polydextrose, and (vii) between about 1×10⁵ cfu/100 kcalsto about 1.5×10¹⁰ cfu/100 kcals of Lactobacillus rhamnosus GG or about1×10⁵ cell equivalent/100 kcals to about 1.5×10¹⁰ cell equivalent/100kcals of dry composition of Lactobacillus rhamnosus GG. In someembodiments, the nutritional composition comprises the culturesupernatant from about 0.015 g per 100 kcal to about 1.5 g per 100 kcal.

The disclosed nutritional composition(s) may be provided in any formknown in the art, such as a powder, a gel, a suspension, a paste, asolid, a liquid, a liquid concentrate, a reconstitutable powdered milksubstitute or a ready-to-use product. The nutritional composition may,in certain embodiments, comprise a nutritional supplement, children'snutritional product, infant formula, human milk fortifier, growing-upmilk or any other nutritional composition designed for a pediatricsubject. Nutritional compositions of the present disclosure include, forexample, orally-ingestible, health-promoting substances including, forexample, foods, beverages, tablets, capsules and powders. Moreover, thenutritional composition of the present disclosure may be standardized toa specific caloric content, it may be provided as a ready-to-useproduct, or it may be provided in a concentrated form. In someembodiments, the nutritional composition is in powder form with aparticle size in the range of 5 μm to 1500 μm, more preferably in therange of 10 μm to 1000 μm, and even more preferably in the range of 50μm to 300 μm.

In some embodiments, the nutritional composition is an infant formulasuitable for infants ranging in age from 0 to 12 months, from 0 to 3months, 0 to 6 months or 6 to 12 months. In other embodiments, thedisclosure provides a fortified milk-based growing-up milk designed forchildren ages 1-3 years and/or 4-6 years, wherein the growing-up milksupports growth and development and life-long health.

As noted, the nutritional composition(s) of the disclosure may comprisea protein source. The protein source can be any used in the art, e.g.,nonfat milk, whey protein, casein, soy protein, hydrolyzed protein,amino acids, and the like. Bovine milk protein sources useful inpracticing the present disclosure include, but are not limited to, milkprotein powders, milk protein concentrates, milk protein isolates,nonfat milk solids, nonfat milk, nonfat dry milk, whey protein, wheyprotein isolates, whey protein concentrates, sweet whey, acid whey,casein, acid casein, caseinate (e.g. sodium caseinate, sodium calciumcaseinate, calcium caseinate) and any combinations thereof.

In one embodiment, the hydrolysates of the present disclosure areprovided without an additional protein source. In other embodiments, theproteins are provided as a combination of both intact proteins and thehydrolyzed proteins disclosed herein. In still other embodiments, thehydrolysates of the present disclosure are provided in combination withamino acids. In yet another embodiment, the hydrolysates of the presentdisclosure may be supplemented with glutamine-containing peptides.

In a particular embodiment of the nutritional composition, thewhey:casein ratio of the protein source is similar to that found inhuman breast milk. In an embodiment, the protein source comprises fromabout 40% to about 90% whey protein and from about 10% to about 60%casein.

In some embodiments, the nutritional composition comprises between about1 g and about 7 g of a protein source per 100 kcal. In otherembodiments, the nutritional composition comprises between about 3.5 gand about 4.5 g of protein per 100 kcal.

In some embodiments, the protein equivalent source comprises ahydrolyzed protein, which includes partially hydrolyzed protein andextensively hydrolyzed protein, such as casein. In some embodiments, theprotein equivalent source comprises a hydrolyzed protein includingpeptides having a molar mass distribution of greater than 500 Daltons.In some embodiments, the hydrolyzed protein comprises peptides, whereinat least 80% of the peptide have a molar mass distribution in the rangeof from about 500 Daltons to about 2,500 Daltons. Still, in someembodiments, the hydrolyzed protein comprises peptides wherein at least80% of the peptides have a molar mass distribution range of from about500 Daltons to about 5,000 Daltons.

In some embodiments, the nutritional composition comprises between about1 g and about 7 g of a protein equivalent source per 100 Kcal. In otherembodiments, the nutritional composition comprises between about 3.5 gand about 4.5 g of protein equivalent source per 100 Kcal.

In certain embodiments, the protein equivalent source comprises aminoacids and is substantially free of whole, intact protein. In certainembodiments, the protein equivalent source includes from about 10% toabout 90% w/w of essential amino acids based on the total amino acidsincluded in the protein equivalent source. In certain embodiments, theprotein equivalent source includes from about 25% to about 75% w/w ofessential amino acids based on the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 40% to about 60% of essential amino acidsbased on the total amino acids included in the protein equivalentsource.

In some embodiments, the protein equivalent source includesnon-essential amino acids. In certain embodiments, the proteinequivalent source includes from about 10% to about 90% w/w ofnon-essential amino acids based on the total amino acids included in theprotein equivalent source. In certain embodiments, the proteinequivalent source includes from about 25% to about 75% w/w ofnon-essential amino acids based on the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 40% to about 60% w/w of non-essential aminoacids based on the total amino acids included in the protein equivalentsource.

In some embodiments, the protein equivalent source includes leucine. Insome embodiments, the protein equivalent source includes from about 2%to about 15% w/w leucine per the total amount of amino acids included inthe protein equivalent source. In some embodiments, the proteinequivalent source includes from about 4% to about 10% w/w leucine perthe total amount of amino acids included in the protein equivalentsource.

In some embodiments, the protein equivalent source includes lysine. Insome embodiments, the protein equivalent source includes from about 2%to about 10% w/w lysine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 4% to about 8% w/w lysine per the total aminoacids in the protein equivalent source.

In some embodiments, the protein equivalent source includes valine. Insome embodiments, the protein equivalent source includes from about 2%to about 15% w/w valine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 4% to about 10% w/w valine per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes isoleucine.In some embodiments, the protein equivalent source includes from about1% to about 8% w/w isoleucine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 3% to about 7% w/w isoleucine per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes threonine.In some embodiments, the protein equivalent source includes from about1% to about 8% w/w threonine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 3% to about 7% w/w threonine per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes tyrosine. Insome embodiments, the protein equivalent source includes from about 1%to about 8% w/w tyrosine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 3% to about 7% w/w tyrosine per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includesphenylalanine. In some embodiments, the protein equivalent sourceincludes from about 1% to about 8% w/w phenylalanine per the total aminoacids included in the protein equivalent source. In some embodiments,the protein equivalent source includes from about 3% to about 7% w/wphenylalanine per the total amino acids in the protein equivalentsource.

In some embodiments, the protein equivalent source includes histidine.In some embodiments, the protein equivalent source includes from about0.5% to about 4% w/w histidine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 1.5% to about 3.5% w/w histidine per thetotal amino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes cysteine. Insome embodiments, the protein equivalent source includes from about 0.5%to about 4% w/w cysteine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 1.5% to about 3.5% w/w cysteine per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes tryptophan.In some embodiments, the protein equivalent source includes from about0.5% to about 4% w/w tryptophan per the total amino acids included inthe protein equivalent source. In some embodiments, the proteinequivalent source includes from about 1.5% to about 3.5% w/w tryptophanper the total amino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes methionine.In some embodiments, the protein equivalent source includes from about0.5% to about 4% w/w methionine per the total amino acids included inthe protein equivalent source. In some embodiments, the proteinequivalent source includes from about 1.5% to about 3.5% w/w methionineper the total amino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes asparticacid. In some embodiments, the protein equivalent source includes fromabout 7% to about 20% w/w aspartic acid per the total amino acidsincluded in the protein equivalent source. In some embodiments, theprotein equivalent source includes from about 10% to about 17% w/waspartic acid per the total amino acids in the protein equivalentsource.

In some embodiments, the protein equivalent source includes proline. Insome embodiments, the protein equivalent source includes from about 5%to about 12% w/w proline per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 7% to about 10% w/w proline per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes alanine. Insome embodiments, the protein equivalent source includes from about 3%to about 10% w/w alanine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 5% to about 8% w/w alanine per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes glutamate.In some embodiments, the protein equivalent source includes from about1.5% to about 8% w/w glutamate per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 3% to about 6% w/w glutamate per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes serine. Insome embodiments, the protein equivalent source includes from about 1.5%to about 8% w/w serine per the total amino acids included in the proteinequivalent source. In some embodiments, the protein equivalent sourceincludes from about 3% to about 5% w/w serine per the total amino acidsin the protein equivalent source.

In some embodiments, the protein equivalent source includes arginine. Insome embodiments, the protein equivalent source includes from about 2%to about 8% w/w arginine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 3.5% to about 6% w/w arginine per the totalamino acids in the protein equivalent source.

In some embodiments, the protein equivalent source includes glycine. Insome embodiments, the protein equivalent source includes from about 0.5%to about 6% w/w glycine per the total amino acids included in theprotein equivalent source. In some embodiments, the protein equivalentsource includes from about 1.5% to about 3.5% w/w glycine per the totalamino acids in the protein equivalent source.

In some embodiments, the nutritional composition comprises between about1 g and about 7 g of a protein equivalent source per 100 Kcal. In otherembodiments, the nutritional composition comprises between about 3.5 gand about 4.5 g of protein equivalent source per 100 Kcal.

In some embodiments, the nutritional composition comprises between about0.5 g/100 Kcal and about 2.5 g/100 Kcal of essential amino acids. Incertain embodiments, the nutritional composition comprises between about1.3 g/100 Kcal to about 1.6 Kcal of essential amino acids.

In some embodiments, the nutritional composition comprises between about0.5 g/100 Kcal and about 2.5 g/100 Kcal of essential amino acids. Incertain embodiments, the nutritional composition comprises between about1.3 g/100 Kcal to about 1.6 Kcal of non-essential amino acids.

In some embodiments, the nutritional composition comprises from about0.2 g/100 Kcal to about 0.5 g/100 Kcal of leucine. In some embodiments,the nutritional composition comprises from about 0.1 g/100 Kcal to about0.4 g/100 Kcal of lysine. In some embodiments, the nutritionalcomposition comprises from about 0.1 g/100 Kcal to about 0.4 g/100 Kcalof valine. In some embodiments, the nutritional composition comprisesfrom about 0.08 g/100 Kcal to about 0.23 g/100 Kcal of isoleucine. Insome embodiments, the nutritional composition comprises from about 0.08g/100 Kcal to about 0.20 g/100 Kcal of threonine. In some embodiments,the nutritional composition comprises from about 0.10 g/100 Kcal toabout 0.15 g/100 Kcal of tyrosine. In some embodiments, the nutritionalcomposition comprises from about 0.05 g/100 Kcal to about 0.15 g/100Kcal of phenylalanine. In some embodiments, the nutritional compositioncomprises from about 0.01 g/100 Kcal to about 0.09 g/100 Kcal ofhistidine. In some embodiments, the nutritional composition comprisesfrom about 0.02 g/100 Kcal to about 0.08 g/100 Kcal of cysteine. In someembodiments, the nutritional composition comprises from about 0.02 g/100Kcal to about 0.08 g/100 Kcal of tryptophan. In some embodiments, thenutritional composition comprises from about 0.02 g/100 Kcal to about0.08 g/100 Kcal of methionine.

In some embodiments, the nutritional composition comprises from about0.2 g/100 Kcal to about 0.7 g/100 Kcal of aspartic acid. In someembodiments, the nutritional composition comprises from about 0.1 g/100Kcal to about 0.4 g/100 Kcal of proline. In some embodiments, thenutritional composition comprises from about 0.1 g/100 Kcal to about 0.3g/100 Kcal of alanine. In some embodiments, the nutritional compositioncomprises from about 0.08 g/100 Kcal to about 0.25 g/100 Kcal ofglutamate. In some embodiments, the nutritional composition comprisesfrom about 0.08 g/100 Kcal to about 0.2 g/100 Kcal of serine. In someembodiments, the nutritional composition comprises from about 0.08 g/100Kcal to about 0.15 g/100 Kcal of arginine. In some embodiments, thenutritional composition comprises from about 0.02 g/100 Kcal to about0.08 g/100 Kcal of glycine.

The nutritional composition(s) of the present disclosure including theprotein equivalent source, may be administered in one or more dosesdaily. Any orally acceptable dosage form is contemplated by the presentdisclosure. Examples of such dosage forms include, but are not limitedto pills, tablets, capsules, soft-gels, liquids, liquid concentrates,powders, elixirs, solutions, suspensions, emulsions, lozenges, beads,cachets, and combinations thereof.

In some embodiments, the protein equivalent source may provide fromabout 5% to about 20% of the total calories for the nutritionalcomposition. In some embodiments, the protein equivalent source mayprovide from about 8% to about 12% of the total calories for thenutritional composition.

Carbohydrate sources can be any used in the art, e.g., lactose, glucose,fructose, corn syrup solids, maltodextrins, sucrose, starch, rice syrupsolids, and the like. The amount of carbohydrate in the nutritionalcomposition typically can vary from between about 5 g and about 25 g/100kcal. In some embodiments, the nutritional composition comprises betweenabout 3 g and about 8 g of a carbohydrate source.

Carbohydrate sources can be any used in the art, e.g., lactose, glucose,fructose, corn syrup solids, maltodextrins, sucrose, starch, rice syrupsolids, and the like. The amount of carbohydrate in the nutritionalcomposition typically can vary from between about 5 g and about 25 g/100Kcal. In some embodiments, the amount of carbohydrate is between about 6g and about 22 g/100 Kcal. In other embodiments, the amount ofcarbohydrate is between about 12 g and about 14 g/100 Kcal. In someembodiments, corn syrup solids are preferred. Moreover, hydrolyzed,partially hydrolyzed, and/or extensively hydrolyzed carbohydrates may bedesirable for inclusion in the nutritional composition due to their easydigestibility. Specifically, hydrolyzed carbohydrates are less likely tocontain allergenic epitopes.

Suitable fat or lipid sources for the nutritional composition of thepresent disclosure may be any known or used in the art, including butnot limited to, animal sources, e.g., milk fat, butter, butter fat, eggyolk lipid; marine sources, such as fish oils, marine oils, single celloils; vegetable and plant oils, such as corn oil, canola oil, sunfloweroil, soybean oil, palm olein oil, coconut oil, high oleic sunflower oil,evening primrose oil, rapeseed oil, olive oil, flaxseed (linseed) oil,cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil,wheat germ oil; medium chain triglyceride oils and emulsions and estersof fatty acids; and any combinations thereof.

In some embodiments, the nutritional composition comprises between about1 g and about 10 g per 100 kcal of a lipid source. In some embodiments,the nutritional composition comprises between about 2 g/100 Kcal toabout 7 g/100 Kcal of a fat source. In other embodiments the fat sourcemay be present in an amount from about 2.5 g/100 Kcal to about 6 g/100Kcal. In still other embodiments, the fat source may be present in thenutritional composition in an amount from about 3 g/100 Kcal to about 4g/100 Kcal. In some embodiments, the nutritional composition comprisesbetween about 3 g and about 8 g per 100 kcal of a lipid source. In someembodiments, the nutritional composition comprises between about 5 andabout 6 g per 100 kcal of a lipid source.

In some embodiments, the fat or lipid source comprises from about 10% toabout 35% palm oil per the total amount of fat or lipid. In someembodiments, the fat or lipid source comprises from about 15% to about30% palm oil per the total amount of fat or lipid. Yet in otherembodiments, the fat or lipid source may comprise from about 18% toabout 25% palm oil per the total amount of fat or lipid.

In certain embodiments, the fat or lipid source may be formulated toinclude from about 2% to about 16% soybean oil based on the total amountof fat or lipid. In some embodiments, the fat or lipid source may beformulated to include from about 4% to about 12% soybean oil based onthe total amount of fat or lipid. In some embodiments, the fat or lipidsource may be formulated to include from about 6% to about 10% soybeanoil based on the total amount of fat or lipid.

In certain embodiments, the fat or lipid source may be formulated toinclude from about 2% to about 16% coconut oil based on the total amountof fat or lipid. In some embodiments, the fat or lipid source may beformulated to include from about 4% to about 12% coconut oil based onthe total amount of fat or lipid. In some embodiments, the fat or lipidsource may be formulated to include from about 6% to about 10% coconutoil based on the total amount of fat or lipid.

In certain embodiments, the fat or lipid source may be formulated toinclude from about 2% to about 16% sunflower oil based on the totalamount of fat or lipid. In some embodiments, the fat or lipid source maybe formulated to include from about 4% to about 12% sunflower oil basedon the total amount of fat or lipid. In some embodiments, the fat orlipid source may be formulated to include from about 6% to about 10%sunflower oil based on the total amount of fat or lipid.

In some embodiments, the oils, i.e. sunflower oil, soybean oil,sunflower oil, palm oil, etc. are meant to cover fortified versions ofsuch oils known in the art. For example, in certain embodiments, the useof sunflower oil may include high oleic sunflower oil. In otherexamples, the use of such oils may be fortified with certain fattyacids, as known in the art, and may be used in the fat or lipid sourcedisclosed herein.

In some embodiments, the fat or lipid source includes an oil blendincluding sunflower oil, medium chain triglyceride oil, and soybean oil.In some embodiments, the fat or lipid source includes a ratio ofsunflower oil to medium chain triglyceride oil of about 1:1 to about2:1. In certain other embodiments, the fat or lipid source includes aratio of sunflower oil to soybean oil of from about 1:1 to about 2:1. Instill other embodiments, the fat or lipid source may include a ratio ofmedium chain triglyceride oil to soybean oil of from about 1:1 to about2:1.

In certain embodiments the fat or lipid source may comprise from about15% to about 50% w/w sunflower oil based on the total fat or lipidcontent. In certain embodiments, the fat or lipid source includes fromabout 25% to about 40% w/w sunflower oil based on the total fat or lipidcontent. In some embodiments, the fat or lipid source comprises fromabout 30% to about 35% w/w sunflower oil based on the total fat or lipidcontent.

In certain embodiments the fat or lipid source may comprise from about15% to about 50% w/w medium chain triglyceride oil based on the totalfat or lipid content. In certain embodiments, the fat or lipid sourceincludes from about 25% to about 40% w/w medium chain triglyceride oilbased on the total fat or lipid content. In some embodiments, the fat orlipid source comprises from about 30% to about 35% w/w medium chaintriglyceride oil based on the total fat or lipid content.

In certain embodiments the fat or lipid source may comprise from about15% to about 50% w/w soybean oil based on the total fat or lipidcontent. In certain embodiments, the fat or lipid source includes fromabout 25% to about 40% w/w soybean oil based on the total fat or lipidcontent. In some embodiments, the fat or lipid source comprises fromabout 30% to about 35% w/w soybean oil based on the total fat or lipidcontent.

In some embodiments, the nutritional composition comprises from about 1g/100 Kcal to about 3 g/100 Kcal of sunflower oil. In some embodiments,the nutritional composition comprises from about 1.3 g/100 Kcal to about2.5 g/100 Kcal of sunflower oil. In still other embodiments, thenutritional composition comprises from about 1.7 g/100 Kcal to about 2.1g/100 Kcal of sunflower oil. The sunflower oil as described herein may,in some embodiments, include high oleic sunflower oil.

In certain embodiments, the nutritional composition if formulated toinclude from about 1 g/100 Kcal to about 2.5 g/100 Kcal of medium chaintriglyceride oil. In other embodiments, the nutritional compositionincludes from about 1.3 g/100 Kcal to about 2.1 g/100 Kcal of mediumchain triglyceride oil. Still in further embodiments, the nutritionalcomposition includes from about 1.6 g/100 Kcal to about 1.9 g/100 Kcalof medium chain triglyceride oil.

In some embodiments, the nutritional composition may be formulated toinclude from about 1 g/100 Kcal to about 2.3 g/100 Kcal of soybean oil.In certain embodiments, the nutritional composition may be formulated toinclude from about 1.2 g/100 Kcal to about 2 g/100 Kcal of soybean oil.Still in certain embodiments, the nutritional composition may beformulated to include from about 1.5 g/100 Kcal to about 1.8 g/100 Kcalof soybean oil.

In some embodiments, the term “sunflower oil”, “medium chaintriglyceride oil”, and “soybean oil” are meant to cover fortifiedversions of such oils known in the art. For example, in certainembodiments, the use of sunflower oil may include high oleic sunfloweroil. In other examples, the use of such oils may be fortified withcertain fatty acids, as known in the art, and may be used in the fat orlipid source disclosed herein.

In some embodiments, the fat or lipid source provides from about 35% toabout 55% of the total calories of the nutritional composition. In otherembodiments, the fat or lipid source provides from about 40% to about47% of the total calories of the nutritional composition.

In certain embodiments the nutritional composition may be formulatedsuch that from about 10% to about 23% of the total calories of thenutritional composition are provided by sunflower oil. In otherembodiments, from about 13% to about 20% of the total calories in thenutritional composition may be provided by sunflower oil. Still, inother embodiments, from about 15% to about 18% of the total calories ofthe nutritional composition may be provided by sunflower oil.

In some embodiments, the nutritional composition may be formulated suchthat from about 10% to about 20% of the total calories are provided byMCT oil. In certain embodiments, from about 12% to about 18% of thetotal calories in the nutritional composition may be provided by MCToil. Still, in certain embodiments, from about 14% to about 17% of thecalories of the nutritional composition may be provided by MCT oil.

In some embodiments, the nutritional composition may be formulated suchthat from about 10% to 20% of the total calories of the nutritionalcomposition are provided by soybean oil. In certain embodiments, fromabout 12% to about 18% of the total calories of the nutritionalcomposition may be provided by soybean oil. In certain embodiments, fromabout 13% to about 16% of the total calories may be provided by soybeanoil.

The nutritional composition of the present disclosure may compriselactoferrin in some embodiments. Lactoferrins are single chainpolypeptides of about 80 kD containing 1-4 glycans, depending on thespecies. The 3-D structures of lactoferrin of different species are verysimilar, but not identical. Each lactoferrin comprises two homologouslobes, called the N- and C-lobes, referring to the N-terminal andC-terminal part of the molecule, respectively. Each lobe furtherconsists of two sub-lobes or domains, which form a cleft where theferric ion (Fe3+) is tightly bound in synergistic cooperation with a(bi)carbonate anion. These domains are called N1, N2, C1 and C2,respectively. The N-terminus of lactoferrin has strong cationic peptideregions that are responsible for a number of important bindingcharacteristics. Lactoferrin has a very high isoelectric point (˜pl 9)and its cationic nature plays a major role in its ability to defendagainst bacterial, viral, and fungal pathogens. There are severalclusters of cationic amino acids residues within the N-terminal regionof lactoferrin mediating the biological activities of lactoferrinagainst a wide range of microorganisms.

Lactoferrin for use in the present disclosure may be, for example,isolated from the milk of a non-human animal or produced by agenetically modified organism. The nutritional compositions describedherein can, in some embodiments comprise non-human lactoferrin,non-human lactoferrin produced by a genetically modified organism and/orhuman lactoferrin produced by a genetically modified organism.

Suitable non-human lactoferrins for use in the present disclosureinclude, but are not limited to, those having at least 48% homology withthe amino acid sequence of human lactoferrin. For instance, bovinelactoferrin (“bLF”) has an amino acid composition which has about 70%sequence homology to that of human lactoferrin. In some embodiments, thenon-human lactoferrin has at least 65% homology with human lactoferrinand in some embodiments, at least 75% homology. Non-human lactoferrinsacceptable for use in the present disclosure include, withoutlimitation, bLF, porcine lactoferrin, equine lactoferrin, buffalolactoferrin, goat lactoferrin, murine lactoferrin and camel lactoferrin.

In some embodiments, the nutritional composition of the presentdisclosure comprises non-human lactoferrin, for example bovinelactoferrin (bLF). bLF is a glycoprotein that belongs to the irontransporter or transferrin family. It is isolated from bovine milk,wherein it is found as a component of whey. There are known differencesbetween the amino acid sequence, glycosylation patters and iron-bindingcapacity in human lactoferrin and bLF. Additionally, there are multipleand sequential processing steps involved in the isolation of bLF fromcow's milk that affect the physiochemical properties of the resultingbLF preparation. Human lactoferrin and bLF are also reported to havedifferences in their abilities to bind the lactoferrin receptor found inthe human intestine.

Though not wishing to be bound by this or any other theory, it isbelieved that bLF isolated from whole milk has less lipopolysaccharide(LPS) initially bound than does bLF that has been isolated from milkpowder. Additionally, it is believed that bLF with a low somatic cellcount has less initially-bound LPS. A bLF with less initially-bound LPShas more binding sites available on its surface. This is thought to aidbLF in binding to the appropriate location and disrupting the infectionprocess.

bLF suitable for the present disclosure may be produced by any methodknown in the art. For example, in U.S. Pat. No. 4,791,193, incorporatedby reference herein in its entirety, Okonogi et al. discloses a processfor producing bovine lactoferrin in high purity. Generally, the processas disclosed includes three steps. Raw milk material is first contactedwith a weakly acidic cationic exchanger to absorb lactoferrin followedby the second step where washing takes place to remove nonabsorbedsubstances. A desorbing step follows where lactoferrin is removed toproduce purified bovine lactoferrin. Other methods may include steps asdescribed in U.S. Pat. Nos. 7,368,141, 5,849,885, 5,919,913 and5,861,491, the disclosures of which are all incorporated by reference intheir entirety.

In certain embodiments, lactoferrin utilized in the present disclosuremay be provided by an expanded bed absorption (“EBA”) process forisolating proteins from milk sources. In particular embodiments, thetarget protein is lactoferrin, though other milk proteins, such aslactoperoxidases or lactalbumins, also may be isolated. The expanded bedadsorption column can be any known in the art, such as those describedin U.S. Pat. Nos. 7,812,138, 6,620,326, and 6,977,046, the disclosuresof which are hereby incorporated by reference herein. EBA technology isfurther described in international published application nos. WO92/00799, WO 02/18237, WO 97/17132, which are hereby incorporated byreference in their entireties.

In other embodiments, lactoferrin for use in the composition of thepresent disclosure can be isolated through the use of radialchromatography or charged membranes, as would be familiar to the skilledartisan.

The lactoferrin that is used in certain embodiments may be anylactoferrin isolated from whole milk and/or having a low somatic cellcount, wherein “low somatic cell count” refers to a somatic cell countless than 200,000 cells/mL. By way of example, suitable lactoferrin isavailable from Tatua Co-operative Dairy Co. Ltd., in Morrinsville, NewZealand, from FrieslandCampina Domo in Amersfoort, Netherlands or fromFonterra Co-Operative Group Limited in Auckland, New Zealand. Thenutritional composition may, in some embodiments, comprise lactoferrinin an amount from about 10 mg/100 mL to about 200 mg/100 mL. In otherembodiments, lactoferrin is present in an amount from about 25 mg/100 mLto about 150 mg/100 mL. In other embodiments lactoferrin is present inan amount from about 60 mg/100 mL to about 120 mg/100 mL. In still otherembodiments lactoferrin is present in an amount from about 85 mg/100 mLto about 110 mg/100 mL.

In certain embodiments lactoferrin is present in an amount of at leastabout 10 mg/100 kcal, at least about 15 mg/100 kcal, at least about 30mg/100 kcal, at least about 50 mg/100 kcal, or at least about 100 mg/100kcal. In certain embodiments, lactoferrin is present in an amount fromabout 10 mg/100 kcal to about 250 mg/100 kcal. In certain embodiments,lactoferrin is present in an amount from about 15 to about 300 mglactoferrin per 100 kcal. In certain embodiments, lactoferrin is presentin an amount from about 50 mg/100 kcal to about 175 mg/100 kcal. Incertain embodiments, lactoferrin is present in an about from about 60mg/100 kcal to about 150 mg per 100 kcal. In yet another embodiment,lactoferrin is present in an about from about 60 mg/100 kcal to about100 mg/100 kcal. In still some embodiments, lactoferrin is present in anamount from about 100 mg/100 kcal to about 150 mg/100 kcals.

In an embodiment, the nutritional composition(s) of the presentdisclosure comprises choline. Choline is a nutrient that is essentialfor normal function of cells. It is a precursor for membranephospholipids, and it accelerates the synthesis and release ofacetylcholine, a neurotransmitter involved in memory storage. Moreover,though not wishing to be bound by this or any other theory, it isbelieved that dietary choline and docosahexaenoic acid (DHA) actsynergistically to promote the biosynthesis of phosphatidylcholine andthus help promote synaptogenesis in human subjects. Additionally,choline and DHA may exhibit the synergistic effect of promotingdendritic spine formation, which is important in the maintenance ofestablished synaptic connections. In some embodiments, the nutritionalcomposition(s) of the present disclosure includes about 40 mg cholineper serving to about 100 mg per 8 oz. serving.

In an embodiment, the nutritional composition comprises a source ofiron. In an embodiment, the source of iron is ferric pyrophosphate,ferric orthophosphate, ferrous fumarate or a mixture thereof and thesource of iron may be encapsulated in some embodiments.

One or more vitamins and/or minerals may also be added in to thenutritional composition in amounts sufficient to supply the dailynutritional requirements of a subject. It is to be understood by one ofordinary skill in the art that vitamin and mineral requirements willvary, for example, based on the age of the subject. For instance, aninfant may have different vitamin and mineral requirements than a childbetween the ages of one and thirteen years. Thus, the embodiments arenot intended to limit the nutritional composition to a particular agegroup but, rather, to provide a range of acceptable vitamin and mineralcomponents.

In certain embodiments, the composition may optionally include, but isnot limited to, one or more of the following vitamins or derivationsthereof: vitamin B1 (thiamin, thiamin pyrophosphate, TPP, thiamintriphosphate, TTP, thiamin hydrochloride, thiamin mononitrate), vitaminB2 (riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide,FAD, lactoflavin, ovoflavin), vitamin B3 (niacin, nicotinic acid,nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD,nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid),vitamin B3-precursor tryptophan, vitamin B6 (pyridoxine, pyridoxal,pyridoxamine, pyridoxine hydrochloride), pantothenic acid (pantothenate,panthenol), folate (folic acid, folacin, pteroylglutamic acid), vitaminB12 (cobalamin, methylcobalamin, deoxyadenosylcobalamin, cyanocobalamin,hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid),vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esterswith other long-chain fatty acids, retinal, retinoic acid, retinolesters), vitamin D (calciferol, cholecalciferol, vitamin D3,1,25,-dihydroxyvitamin D), vitamin E (α-tocopherol, α-tocopherolacetate, α-tocopherol succinate, α-tocopherol nicotinate, α-tocopherol),vitamin K (vitamin K1, phylloquinone, naphthoquinone, vitamin K2,menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8,menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10,menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol,β-carotene and any combinations thereof.

In other embodiments, the composition may optionally include, but is notlimited to, one or more of the following minerals or derivationsthereof: boron, calcium, calcium acetate, calcium gluconate, calciumchloride, calcium lactate, calcium phosphate, calcium sulfate, chloride,chromium, chromium chloride, chromium picolonate, copper, coppersulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyliron, ferric iron, ferrous fumarate, ferric orthophosphate, irontrituration, polysaccharide iron, iodide, iodine, magnesium, magnesiumcarbonate, magnesium hydroxide, magnesium oxide, magnesium stearate,magnesium sulfate, manganese, molybdenum, phosphorus, potassium,potassium phosphate, potassium iodide, potassium chloride, potassiumacetate, selenium, sulfur, sodium, docusate sodium, sodium chloride,sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate andmixtures thereof. Non-limiting exemplary derivatives of mineralcompounds include salts, alkaline salts, esters and chelates of anymineral compound.

The minerals can be added to growing-up milks or to other children'snutritional compositions in the form of salts such as calcium phosphate,calcium glycerol phosphate, sodium citrate, potassium chloride,potassium phosphate, magnesium phosphate, ferrous sulfate, zinc sulfate,cupric sulfate, manganese sulfate, and sodium selenite. Additionalvitamins and minerals can be added as known within the art.

In an embodiment, the children's nutritional composition may containbetween about 10 and about 50% of the maximum dietary recommendation forany given country, or between about 10 and about 50% of the averagedietary recommendation for a group of countries, per serving of vitaminsA, C, and E, zinc, iron, iodine, selenium, and choline. In anotherembodiment, the children's nutritional composition may supply about10-30% of the maximum dietary recommendation for any given country, orabout 10-30% of the average dietary recommendation for a group ofcountries, per serving of B-vitamins. In yet another embodiment, thelevels of vitamin D, calcium, magnesium, phosphorus, and potassium inthe children's nutritional product may correspond with the averagelevels found in milk. In other embodiments, other nutrients in thechildren's nutritional composition may be present at about 20% of themaximum dietary recommendation for any given country, or about 20% ofthe average dietary recommendation for a group of countries, perserving.

The children's nutritional composition of the present disclosure mayoptionally include one or more of the following flavoring agents,including, but not limited to, flavored extracts, volatile oils, cocoaor chocolate flavorings, peanut butter flavoring, cookie crumbs, vanillaor any commercially available flavoring. Examples of useful flavoringsinclude, but are not limited to, pure anise extract, imitation bananaextract, imitation cherry extract, chocolate extract, pure lemonextract, pure orange extract, pure peppermint extract, honey, imitationpineapple extract, imitation rum extract, imitation strawberry extract,or vanilla extract; or volatile oils, such as balm oil, bay oil,bergamot oil, cedarwood oil, cherry oil, cinnamon oil, clove oil, orpeppermint oil; peanut butter, chocolate flavoring, vanilla cookiecrumb, butterscotch, toffee, and mixtures thereof. The amounts offlavoring agent can vary greatly depending upon the flavoring agentused. The type and amount of flavoring agent can be selected as is knownin the art.

The nutritional compositions of the present disclosure may optionallyinclude one or more emulsifiers that may be added for stability of thefinal product. Examples of suitable emulsifiers include, but are notlimited to, lecithin (e.g., from egg or soy), alpha lactalbumin and/ormono- and di-glycerides, and mixtures thereof. Other emulsifiers arereadily apparent to the skilled artisan and selection of suitableemulsifier(s) will depend, in part, upon the formulation and finalproduct.

The incorporation of certain ingredients (e.g., HMOs) described hereininto a nutritional composition, such as an infant formula, may requirethe presence of at least one emulsifier to ensure that the ingredientdoes not separate from the fat or proteins contained within the infantformula during shelf-storage or preparation.

In some embodiments, the nutritional composition may be formulated toinclude from about 0.5 wt % to about 1 wt % of emulsifier based on thetotal dry weight of the nutritional composition. In other embodiments,the nutritional composition may be formulated to include from about 0.7wt % to about 1 wt % of emulsifier based on the total dry weight of thenutritional composition.

In some embodiments where the nutritional composition is a ready-to-useliquid composition, the nutritional composition may be formulated toinclude from about 200 mg/L to about 600 mg/L of emulsifier. Still, incertain embodiments, the nutritional composition may include from about300 mg/L to about 500 mg/L of emulsifier. In other embodiments, thenutritional composition may include from about 400 mg/L to about 500mg/L of emulsifier.

The nutritional compositions of the present disclosure may optionallyinclude one or more preservatives that may also be added to extendproduct shelf life. Suitable preservatives include, but are not limitedto, potassium sorbate, sodium sorbate, potassium benzoate, sodiumbenzoate, potassium citrate, calcium disodium EDTA, and mixturesthereof. The incorporation of a preservative in the nutritionalcomposition ensures that the nutritional composition has a suitableshelf-life such that, once reconstituted for administration, thenutritional composition delivers nutrients that are bioavailable and/orprovide health and nutrition benefits for the target subject.

In some embodiments the nutritional composition may be formulated toinclude from about 0.1 wt % to about 1.0 wt % of a preservative based onthe total dry weight of the composition. In other embodiments, thenutritional composition may be formulated to include from about 0.4 wt %to about 0.7 wt % of a preservative based on the total dry weight of thecomposition.

In some embodiments where the nutritional composition is a ready-to-useliquid composition, the nutritional composition may be formulated toinclude from about 0.5 g/L to about 5 g/L of preservative. Still, incertain embodiments, the nutritional composition may include from about1 g/L to about 3 g/L of preservative.

The nutritional compositions of the present disclosure may optionallyinclude one or more stabilizers. Suitable stabilizers for use inpracticing the nutritional composition of the present disclosureinclude, but are not limited to, gum arabic, gum ghatti, gum karaya, gumtragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum,pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC(sodium carboxymethylcellulose), methylcellulose hydroxypropyl methylcellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid estersof mono- and diglycerides), dextran, carrageenan, and mixtures thereof.

Incorporating a suitable stabilizer in the nutritional compositionensures that the nutritional composition has a suitable shelf-life suchthat, once reconstituted for administration, the nutritional compositiondelivers nutrients that are bioavailable and/or provide health andnutrition benefits for the target subject.

In some embodiments where the nutritional composition is a ready-to-useliquid composition, the nutritional composition may be formulated toinclude from about 50 mg/L to about 150 mg/L of stabilizer. Still, incertain embodiments, the nutritional composition may include from about80 mg/L to about 120 mg/L of stabilizer.

The nutritional compositions of the disclosure may provide minimal,partial or total nutritional support. The compositions may benutritional supplements or meal replacements. The compositions may, butneed not, be nutritionally complete. In an embodiment, the nutritionalcomposition of the disclosure is nutritionally complete and containssuitable types and amounts of lipid, carbohydrate, protein, vitamins andminerals. The amount of lipid or fat typically can vary from about 2 toabout 7 g/100 kcal. The amount of protein typically can vary from about1 to about 5 g/100 kcal. The amount of carbohydrate typically can varyfrom about 8 to about 14 g/100 kcal.

In some embodiments, the nutritional composition of the presentdisclosure is a growing-up milk. Growing-up milks are fortifiedmilk-based beverages intended for children over 1 year of age (typicallyfrom 1-6 years of age). They are not medical foods and are not intendedas a meal replacement or a supplement to address a particularnutritional deficiency. Instead, growing-up milks are designed with theintent to serve as a complement to a diverse diet to provide additionalinsurance that a child achieves continual, daily intake of all essentialvitamins and minerals, macronutrients plus additional functional dietarycomponents, such as non-essential nutrients that have purportedhealth-promoting properties.

The exact composition of an infant formula or a growing-up milk or othernutritional composition according to the present disclosure can varyfrom market-to-market, depending on local regulations and dietary intakeinformation of the population of interest. In some embodiments,nutritional compositions according to the disclosure consist of a milkprotein source, such as whole or skim milk, plus added sugar andsweeteners to achieve desired sensory properties, and added vitamins andminerals. The fat composition is typically derived from the milk rawmaterials. Total protein can be targeted to match that of human milk,cow milk or a lower value. Total carbohydrate is usually targeted toprovide as little added sugar, such as sucrose or fructose, as possibleto achieve an acceptable taste. Typically, Vitamin A, calcium andVitamin D are added at levels to match the nutrient contribution ofregional cow milk. Otherwise, in some embodiments, vitamins and mineralscan be added at levels that provide approximately 20% of the dietaryreference intake (DRI) or 20% of the Daily Value (DV) per serving.Moreover, nutrient values can vary between markets depending on theidentified nutritional needs of the intended population, raw materialcontributions and regional regulations.

The pediatric subject may be a child or an infant. For example, thesubject may an infant ranging in age from 0 to 3 months, about 0 to 6months, 0 to 12 months, 3 to 6 months, or 6 to 12 months. The subjectmay alternatively be a child ranging in age from 1 to 13 years, 1 to 6years or 1 to 3 years. In an embodiment, the composition may beadministered to the pediatric subject prenatally, during infancy, andduring childhood.

EXAMPLE Example 1

This example illustrates a method for producing a partially hydrolyzedcasein which is enriched with beta-casein protein. Beta-casein enrichedcasein protein is dispersed in water with temperature about 55° C. ThepH of the slurry is adjusted with sodium and/or potassium hydroxide tothe range from about 6 to 9, preferably 7.0. The enzyme of trypsin andchymotrypsin, or trypsin-like and chymotrypsin-like enzymes from animaland/or microbial sources are added into the slurry. The incubation timemay be from 2 to 6 hours. At the end of digestion, the slurry is heattreated to inactivate the enzymes (e.g., 10 minutes at temperature of85-90° C.). The hydrolysate slurry obtained from the process could befurther centrifuged or filtered to clarify and remove large particlesand protein aggregates, or large protein/peptides molecules ifultrafiltration and/or nanofiltration is used. The hydrolysate could bealso dried to get a powdered product.

Example 2

Table 1 provides an exemplary nutritional formulation comprising anexemplary beta-casein enriched casein hydrolysate as described herein.

TABLE 1 Per Nutrient Unit 100 kcal Cow's milk protein g 1.89 Beta-caseinenriched g 0.21 casein hydrolysate Fat g 5.3 Linoleic Acid mg 810Alpha-Linolenic Acid mg 71 Docosahexaenoic Acid mg 17.8 Arachidonic Acidmg 36 Carbohydrates g 11.2 GOS g 0.31 Polydextrose g 0.31 Vitamin A μg84 Vitamin D μg 1.55 Vitamin E mg 1.27 Vitamin K μg 7.2 Thiamin μg 85Riboflavin μg 170 Vitamin B6 μg 60 Vitamin B12 μg 0.31 Niacin μg 660Folic Acid μg 18 Pantothenic Acid μg 570 Biotin μg 2.7 Vitamin C mg 18Sodium mg 28 Potassium mg 110 Chloride mg 65 Calcium mg 79 Phosphorus mg48 Magnesium mg 8 Iodine μg 17 Iron mg 1 Copper μg 65 Zinc mg 0.8Manganese μg 18 Selenium μg 2.7 Choline mg 24 Inositol mg 8.5 Carnitinemg 2 Taurine mg 6 Total Nucleotides mg 3.1 Lactoferrin g 0.09

Although preferred embodiments of the disclosure have been describedusing specific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the art withoutdeparting from the spirit or the scope of the present disclosure, whichis set forth in the following claims. In addition, it should beunderstood that aspects of the various embodiments may be interchangedin whole or in part. Therefore, the spirit and scope of the appendedclaims should not be limited to the description of the preferredversions contained therein.

All references cited in this specification, including withoutlimitation, all papers, publications, patents, patent applications,presentations, texts, reports, manuscripts, brochures, books, internetpostings, journal articles, periodicals, and the like, are herebyincorporated by reference into this specification in their entireties.The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A method for preparing a protein hydrolysate foruse in a nutritional composition, the method comprising: hydrolyzing amilk protein preparation selected from the group consisting of a casein,acid casein or caseinate, beta-casein enriched casein, acid casein orcaseinate, an alpha-casein enriched casein, acid casein or caseinate,and/or a kappa-casein enriched casein, acid casein or caseinate using aprotease or proteases selected from the group consisting of trypsin or atrypsin-like protease, chymotrypsin or a chymotrypsin-like protease,pepsin and plasmin to form a protein hydrolysate.
 2. The method of claim1, wherein the casein, acid casein or caseinate is hydrolyzed usingtrypsin or trypsin-like protease, chymotrypsin or chymotrypsin-likeprotease, pepsin and plasmin.
 3. The method of claim 1, wherein thecasein, acid casein or caseinate is beta-casein enriched and thebeta-casein enriched casein, acid casein or caseinate comprises betweenabout 40%-95% beta-casein.
 4. The method of claim 1, wherein the casein,acid casein or caseinate is alpha-casein enriched and the alpha-caseinenriched casein, acid casein or caseinate comprises between about50%-95% alpha-casein.
 5. The method of claim 4, wherein the alpha-caseinenriched casein, acid casein or caseinate further comprises betweenabout 15%-95% kappa-casein.
 6. The method of claim 1, wherein thecasein, acid casein or caseinate is kappa-casein enriched and thekappa-enriched casein, acid casein or caseinate comprises between about15-95% kappa-casein.
 7. The method of claim 1, further comprisinghydrolyzing a protein selected from the group consisting of polymericimmunoglobulin receptor (PIGR), osteopontin, bile-salt activated lipase,and clusterin using a protease selected from the group consisting oftrypsin, chymotrypsin and plasmin.
 8. A method for preparing anutritional composition, the method comprising: hydrolyzing a milkprotein preparation selected from the group consisting of a casein, acidcasein or caseinate, beta-casein enriched casein, acid casein orcaseinate, an alpha-casein enriched casein, acid casein or caseinate,and/or a kappa-casein enriched casein, acid casein or caseinate using aprotease selected from the group consisting of trypsin or trypsin-likeprotease, chymotrypsin or chymotrypsin-like protease, pepsin and plasminto form a protein hydrolysate, where in the protein hydrolysate isincluded in the nutritional composition.
 9. The method of claim 8,wherein the nutritional composition is a pediatric nutritionalcomposition.
 10. The method of claim 9, wherein the pediatricnutritional composition is an infant formula.
 11. The method of claim 8,wherein the nutritional composition further comprises: (i) a proteinsource, (ii) a lipid source, and/or (iii) a carbohydrate source.
 12. Themethod of claim 11, wherein the nutritional composition furthercomprises (iv) a prebiotic, and/or (vi) a probiotic.
 13. The method ofclaim 12, wherein the probiotic comprises a Lactobacillus species. 14.The method of claim 13, wherein the probiotic comprises Lactobacillusrhamnosus GG.
 15. The method of claim 12, wherein the probiotic isnon-viable.
 16. The method of claim 12, wherein the probiotic is viable.17. The method of claim 12, wherein the probiotic is present in anamount ranging from about 1×10⁵ cfu/100 kcals to about 1.5×10⁹ cfu/100kcals.
 18. The method of claim 8, wherein the composition furthercomprises at least one human milk oligosaccharide (HMO).
 19. The methodof claim 8, further comprising a long chain polyunsaturated fatty acid.20. The method of claim 19, wherein the long chain polyunsaturated fattyacid comprises docosahexaenoic acid and/or arachidonic acid.
 21. Themethod of claim 8, further comprising a source of β-glucan.
 22. Themethod of claim 21, wherein the source of β-glucan comprises aβ-1,3-glucan.
 23. A composition comprising the protein hydrolysate ofclaim 1.